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1.  Does the lipid environment impact the open-state conductance of an engineered β-barrel protein nanopore? 
Biochimica et biophysica acta  2012;1828(3):1057-1065.
Using rational membrane protein design, we were recently able to obtain a β-barrel protein nanopore that was robust under an unusually broad range of experimental circumstances. This protein nanopore was based upon the native scaffold of the bacterial ferric hydroxamate uptake component A (FhuA) of E. coli. In this work, we expanded the examinations of the open-state current of this engineered protein nanopore, also called FhuA ΔC/Δ4L, employing an array of lipid bilayer systems that contained charged and uncharged as well as conical and cylindrical lipids. Remarkably, systematical single-channel analysis of FhuA ΔC/Δ4L indicated that most of its biophysical features, such as the unitary conductance and the stability of the open-state current, were not altered under the conditions tested in this work. However, electrical recordings at high transmembrane potentials revealed that the presence of conical phospholipids within the bilayer catalyzes the first, stepwise current transition of the FhuA ΔC/Δ4L protein nanopore to a lower-conductance open state. This study reinforces the stability of the open-state current of the engineered FhuA ΔC/Δ4L protein nanopore under various experimental conditions, paving the way for further critical developments in biosensing and molecular biomedical diagnosis.
PMCID: PMC3560310  PMID: 23246446
E. coli FhuA; Planar lipid bilayer; Dimensionless shape factor; Channel closure; Single-channel recordings; Lipid effect
2.  Gender disparities in the association between epicardial adipose tissue volume and coronary atherosclerosis: A 3-dimensional cardiac computed tomography imaging study in Japanese subjects 
Growing evidence suggests that epicardial adipose tissue (EAT) may contribute to the development of coronary artery disease (CAD). In this study, we explored gender disparities in EAT volume (EATV) and its impact on coronary atherosclerosis.
The study population consisted of 90 consecutive subjects (age: 63 ± 12 years; men: 47, women: 43) who underwent 256-slice multi-detector computed tomography (MDCT) coronary angiography. EATV was measured as the sum of cross-sectional epicardial fat area on CT images, from the lower surface of the left pulmonary artery origin to the apex. Subjects were segregated into the CAD group (coronary luminal narrowing > 50%) and non-CAD group.
EATV/body surface area (BSA) was higher among men in the CAD group than in the non-CAD group (62 ± 13 vs. 33 ± 10 cm3/m2, p < 0.0001), but did not differ significantly among women in the 2 groups (49 ± 18 vs. 42 ± 9 cm3/m2, not significant). Multivariate logistic analysis showed that EATV/BSA was the single predictor for >50% coronary luminal narrowing in men (p < 0.0001). Predictors excluded were age, body mass index, hypertension, diabetes mellitus, and hyperlipidemia.
Increased EATV is strongly associated with coronary atherosclerosis in men.
PMCID: PMC3489699  PMID: 22963346
Atherosclerosis; Gender difference; Epicardial adipose tissue; Obesity
3.  Diagnostic utility of cardiac magnetic resonance for detection of cardiac involvement in female carriers of Duchenne muscular dystrophy 
Heart Asia  2010;2(1):52-55.
Cardiac involvement is a recognised complication in female carriers of Duchenne muscular dystrophy (DMD). Since segmental or global left ventricle (LV) wall motion abnormalities in DMD carriers can arise even without apparent muscle weakness, it is difficult to differentiate cardiac involvement of a DMD carrier from other heart diseases in a non-invasive manner. Cardiac magnetic resonance (CMR) with late gadolinium enhancement (LGE) enables assessment of regional wall motion abnormality and myocardial damage with high spatial resolution.
To assess the utility of CMR for detection of myocardial damage in female carriers of DMD.
Methods and results
Gadolinium-enhanced CMR was performed in seven female DMD carriers. Physical examination, electrocardiography, chest radiograph, measurements of total creatinine kinase and brain natriuretic peptide levels, and two-dimensional echocardiography were also performed. Four (57%) of the seven carriers had LGE, and LGE was frequently observed at the subepicardial layer in the inferolateral segment. Two carriers had a focal LGE at the LV inferolateral wall without LV dilation or wall motion abnormalities.
CMR findings of DMD carriers were characterised by subepicardial LGE, which was localised at inferolateral segments. CMR may be a useful modality for detecting cardiac involvement in DMD carriers.
PMCID: PMC4898527  PMID: 27325943
MRI; gadolinium; muscular dystrophies; cardiomyopathies
4.  Cloning, Expression, and Cell Surface Localization of Paenibacillus sp. Strain W-61 Xylanase 5, a Multidomain Xylanase 
Applied and Environmental Microbiology  2003;69(12):6969-6978.
We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.
PMCID: PMC310030  PMID: 14660338
5.  Stochastic Assembly of Two-Component Staphylococcal γ-Hemolysin into Heteroheptameric Transmembrane Pores with Alternate Subunit Arrangements in Ratios of 3:4 and 4:3 
Journal of Bacteriology  2002;184(17):4747-4756.
Self-assembling, pore-forming toxins from Staphylococcus aureus are illustrative molecules for the study of the assembly and membrane insertion of oligomeric transmembrane proteins. On the basis of previous studies, we have shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of ca. 200 kDa. Here we show that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. High-resolution electron microscopic images of negatively stained pore complexes clearly revealed a heptameric structure. When adjacent monomers in the pore complexes were randomly cross-linked by using glutaraldehyde, LukF-LukF, LukF-Hlg2, and Hlg2-Hlg2 dimers were detected in an approximate ratio of 1:12:1, suggesting that LukF and Hlg2 were alternately arranged in the pore complex in molar ratios of 3:4 and 4:3. The alternate arrangements of LukF and Hlg2 in molar ratios of 3:4 and 4:3 were also visualized under electron microscope with the pore complexes consisting of glutathione S-transferase fusion protein of LukF or Hlg2 and wild-type protein of Hlg2 or LukF, respectively.
PMCID: PMC135295  PMID: 12169599

Results 1-5 (5)